CN1273589C - Conjugation of polypeptides - Google Patents

Conjugation of polypeptides Download PDF

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CN1273589C
CN1273589C CNB971922942A CN97192294A CN1273589C CN 1273589 C CN1273589 C CN 1273589C CN B971922942 A CNB971922942 A CN B971922942A CN 97192294 A CN97192294 A CN 97192294A CN 1273589 C CN1273589 C CN 1273589C
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molecule
conjugate
enzyme
composition
purposes
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CN1211278A (en
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H·比斯加德－弗兰茨
A·A·奥尔森
A·普伦托
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Novozymes AS
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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Abstract

The present invention provides polypeptide conjugates with reduced allergenicity comprising a polymeric carrier molecule having two or more polypeptide molecules coupled thereto. The invention also provides methods for producing the conjugates, compositions comprising the conjugates, and the use of the conjugates in industrial applications, including personal care products and detergent compositions.

Description

Conjugation of polypeptides
The present invention relates to a kind of polypeptide conjugate with the sex change originality that weakens, a kind ofly be used to produce described method with polypeptide conjugate of the sex change originality that weakens, contain described composition with polypeptide conjugate of the allergenicity that weakens, described polypeptide conjugate is used to weaken the purposes of the allergenicity of multiple mechanicals, and is used for purposes such as the multiple purpose of personal-care supplies and detergent composition.
Increasing polypeptide comprises albumen and enzyme, is with commercial run production, to use it in industry, family expenses, foods/feeds, makeup or the medicine at present with microorganism as proteolytic enzyme.Under some occasion, described polypeptide can cause potential harm to being engaged in the personnel that produce the goods contain this peptide species, and can be to a certain extent to the user of this product, damage as the end user of stylist and cosmetic and beautification product etc.
Above-mentioned potential hazard is necessary to be controlled and/or eliminate.
The sex change originality of polypeptide
Generally, polypeptide is a potential antigen, and the human immune system can produce specific antibody after contact.After having obtained useful reaction clinically, this process is called as " immunity ", and if this reaction has caused hypersensitivity, then be referred to as " sensitization ".When contacting first, can cause the colony screening and the amplification of specific B-cell clone, this means the clinical manifestation when protective reaction or transformation reactions will be follow-up the contact.Transformation reactions can be defined as the pathology immune response that the innoxious substance by lower concentration causes.The feature that causes the sensitization process of I type hypersensitivity is to generate special IgE antibody.At present, still not exclusively understand the mechanism that the control hypotype changes.
Excretory IgE can be combined on the Fc epsilon receptor that is positioned at mastocyte and basophil cellular surface by activated B-cell, it contains multiple by kytoplasm particle (J.Klein, " immunology ", the Blackwell Science Press of packing such as the chemical mediator of histamine, London, 1990; E.Benjamini ﹠amp; S.Leskowitz " immunology ", Wiley Liss, N.Y.1991).
In atopic individuals, above-mentioned each cell all can have a large amount of IgE molecules and its surface bonding, and here they still can interact time several weeks with allergen.With allergen contact after, the IgE of described surface bonding can with this allergen cross coupled, can cause the kytoplasm particle release near the described cell, thereby cause the inflammatory transformation reactions.
The effect that had confirmed IgE already is relevant with the innate immune defence system, and described system is at parasitic infection, and has confirmed that allergic is the unfavorable byproduct of this system of defense.
Can classify to causing according to its way of contact: induction type allergen (pollen, dirt mite etc.), absorption type allergen (milk, egg etc.) by the natural allergen of IgE mediation hypersensitivity; Contact-type allergen (as allergen) and from the allergen of biting type insect (as honeybee, fiery ant etc.) from latex.The source of the gas allergen is a maximum clinically class, and outstanding behaviours is the high potentially dangerous field of industrial polypeptide.
Can be undertaken by body internal stimulus method allergic test, modal is the skin penetrating test of being undertaken by multiple in vitro tests, mainly is based on the IgE content in the peripheral blood.Although big quantity research has been made in one field, back, the most reliable method of diagnosis of allergies still is the body internal stimulus, and this method has different sensitivity according to specific target organ.
For example, the nose internal stimulus that carries out with allergenicity albumen can cause transformation reactions, although be negative (IvanRoitt, " basic immunology " the 5th edition of special SERUM IgE when tuerculoderma and radioallergosorbent test (RAST), p.152 and p.240,1984).
Weaken the allergenicity of polypeptide
At present, be by fixing, granulating, coating or dissolve industrial polypeptide product and avoid the transformation reactions that occurs at described goods, particularly avoid the formation of airborne transmission material.In a word, aforesaid method handle and processing during still have and form dust or aerocolloidal danger, and thereby the danger that produces abnormal sensitization.
In a word, still there is the danger of polypeptide dust or dissolved polypeptide that aerosol form occurs.Therefore, the generation of some enzyme release might cause sensitization and consequent transformation reactions.
The another kind of method that overcomes the problems referred to above is to select people source polypeptide, so that produce in such as bacterium, fungi, yeast or mammalian cell cultures.And, can not find people source polypeptide in many instances with ideal behavior, therefore, have to consider other source.Described other source can be human polypeptide, changes to some extent on one or several position of its molecule, to produce required performance.It can also be the molecule from other species, comprises bacterium, mould etc.Each of product of back one class all has cause immunostimulating effectiveness in Mammals.
The another kind of method that is used to reduce allergenicity is to reduce the size of protein molecular (for example, referring to the open No.4 of Japanese Patent, 112,753, or research publication number (Research DisclosureNo.) 335,102).But, this solution only can be used for the unimportant occasion of described proteic activity, or is used for still keeping behind this proteolytic degradation the rare occasion of this protein-active.
Existing people proposes the change by epitope mapping and allergen epi-position subsequently, weakens proteic allergenicity (referring to WO 92/10755 (Novo NordiskA/S)) with protein engineering method.This method need drop into a large amount of work and exploitation usually.
Can be used for weakening described immunity system is " Pegylation " method to the another kind of method of the reaction of polypeptide, and this method comprises by covalent attachment polyoxyethylene glycol (PEG) chain and peptide molecule being linked together modifies polypeptide.This method 20 years just open in the past No. 4,179,337, US patent (for example, referring to), but only be used for medicinal polypeptide now.Therefore, its main purpose is to reduce IgM and/or the IgG that immunity system produced.
Fig. 1 represents by method in the tracheae with showing as the special male Dunkin of enzyme Hartley cavy quantity after 1.0 μ g monomer peroxidase and 1.0 μ g dextran-peroxidase A and the B processing, as the function of all numbers of starting at from processing day.
Fig. 2 represents to handle the quantity that shows as the special male Dunkin of enzyme Hartley cavy afterwards by method in the tracheae with 1.0 μ g lipase and 1.0 μ g dextran-lipase, as the function of all numbers of starting at from processing day.
Fig. 3 represents by method in the tracheae with the quantity that shows as the special male Dunkin of enzyme Hartley cavy after 1.0 μ g cellulases and the 1.0 μ g dextran-cellulose treatment, as the function of all numbers of starting at from processing day.
The purpose of this invention is to provide polypeptide conjugate with the allergenicity that weakens.
Be understandable that with regard to the industrial application of polypeptide, it mainly is the suction allergen that can cause transformation reactions danger.Therefore, one of significant advantage of the present invention is, the inventor has solved the respiratory irritation problem that is caused by allergen, and prior art solutions relates generally to the skin irritation of being undertaken by the immunogen that confirms.Respiratory irritation is a more sensitive issue.
To because these terms can be used often at the immune system response that is used to describe at polypeptide, explain to giving a definition below, even a lot of scientist also is like this for understanding the important several terms of the present invention with unclear form.
Immunogenicity, antigenicity and allergenicity
" immunogenicity " be one than " antigenicity " and " allergenicity " generalized term more, the reaction that the expression immunity system exists allogenic material.According to the immunoreactive type that it caused, respectively described allogenic material is called " immunogen ", " antigen " and " allergen ".
" immunogen " but be a kind of after entering animal and human's the recycle system material of challenge.
" antigen " speech is meant a kind of like this material, and itself can produce antibody after being identified as non-self molecule.
" allergen " is a kind of antigen, and it can cause abnormal sensitization or transformation reactions because of the generation (in human body, and being the molecule with suitable effect) of IgE antibody in animal.
" recycle system " speech of above employed humans and animals body is meant the system that mainly is made up of heart and blood vessel in specification sheets of the present invention.Be used for keeping blood at the required energy of vascular system circulation by the heart supply.The effect of the recycle system is as biological movement system, with O 2, nutritive substance, hormone and other regulate the substance transportation (passing through blood) that plays an important role in tissue for cell.And, by blood with CO 2From tissue, be scavenged into lung, and residual substance is scavenged in the organ such as kidney.In addition, for thermoregulation with comprise the defense mechanism of immunity system, blood plays an important role.
In the context of the present invention, polypeptide conjugate with " allergenicity that weakens " is meant for corresponding parental generation peptide molecule, the IgE that can cause the allergic effect state that is produced when sucking polypeptide conjugate of the present invention (in human body, and being the molecule with suitable effect in particular animals) significantly reduces.
As indicated above, at least in the intended scope of polypeptide of the present invention, be necessary to distinguish by skin contact by the transformation reactions of skin allergen mediation and the transformation reactions that causes by the breathing allergen by contact with cell bonded IgE in the bronchial tree, because well known fact is, even can cause transformation reactions in inhalation test, but tuerculoderma still may be negative.
Therefore, sex change originality is measured and can be finished by inhalation test, relatively uses the parental generation polypeptide and has the effect of the polypeptide of the present invention of the allergenicity that weakens accordingly in the tracheae in test.
The existing multiple external animal model that is used to estimate the polypeptide allergenicity.In these models some can be provided for the suitable basis of human hazard evaluation.Proper model comprises guinea pig model and rat model.These models are attempted to identify the breathing allergen according to the reaction that causes in aforementioned sensitization animal.According to above-mentioned model the allergen of inferring is imported in the animal body by method in the tracheae.
Suitable cavy strain-Dunkin Hartley strain-not as the people, when transformation reactions, to produce IgE.But, they can produce the IgG antibody 1A of another kind of type and IgG1B (for example, referring to Prent φ, ATLA, 19, p.8-14,1991), and above-mentioned antibody is determining the transformation reactions at the suction polypeptide that comprises enzyme.Therefore, when using Dunkin Hartley animal model, the relative quantity of IgG1A and IgG1B is to weigh the yardstick of level of allergenicity.
The rat strain that is suitable for tracheae interior contact polypeptide and enzyme is a Brown Norway strain.Brown Norway product tie up in the transformation reactions and can produce IgE.
Also other animal such as rabbit can be used for comparative study.
Confirm in the surprising discovery of the present invention disclosed in the example 1, for corresponding monomer peroxidase (Mr is approximately 39kDa), (Mr is about 1, and allergenicity 000kDa) has weakened with dextran bonded purifying wild-type Coprinus cinereus (Coprinus cinerea) peroxidase by method introducing in the tracheae.
Example 2 and 3 confirms that the sex change originality of lipase and cellulase has also weakened respectively.
First aspect the present invention relates to a kind of polypeptide conjugate with the allergenicity that weakens, and this conjugate comprises that has two or more polymer support molecules of covalently bound peptide molecule with it.
Can peptide molecule and polymer support molecule be combined with any method.Those skilled in the art know, can be with the chemical groups on the polypeptide, combine as the hydroxyl of free hydroxyl group, free sulfhydryl groups, free acidic group, free amine group and number of polymers (carrier) molecule.
In one embodiment of the present invention, described polymer support molecule combines by formed covalent linkage and two or more peptide molecule between one of two thiazolinyls of divinylsulfone.
In the context of the present invention, " polypeptide " speech is meant the polypeptide of all types, and as albumen, enzyme, part, antibody, inhibitor, acceptor, it can constitute a kind of big or smaller portions of commodity.The special polypeptide of considering is such, and it does not want to make it to enter the recycle system of human body or animal body as a kind of composition of commodity.
Second aspect the present invention relates to the method that a kind of production has the polypeptide of the allergenicity that weakens, and this method may further comprise the steps:
I) activate the polymer support molecule and
Ii) under the condition that is suitable for puting together, allow two or more peptide molecules and the molecular reaction of described activated polymer support and
Iii) seal the residual activity group on the described conjugate.
In a kind of preferred embodiment of the inventive method, described polymer support molecule directly is connected with two or more peptide molecules or connects by active linkers.
The activation of the polymer support molecule step I) can be carried out with any currently known methods in this area.To introduce the example of above-mentioned suitable method of attachment below.
In one embodiment of the present invention, by divinylsulfone two or more peptide molecules and described polymer support molecule are linked together.
The 3rd purpose of the present invention provides the composition that contains polypeptide conjugate of the present invention.
Above-mentioned composition can also comprise polypeptide, as albumen and/or enzyme and/or be usually used in composition in the following products: such as washing composition, household supplies, agrochemicals, personal-care supplies, makeup, cosmetics, oral-cavity article, skin care and hair care articles, be used to handle fabric composition, be used to clean crust composition, be used to produce the composition and the oral and externally applied medicine of food, feed, fruit juice, wine and beverage.
The above-mentioned type product is known as " Industrial products " sometimes.Hereinafter, this term is used to the special series products of considering of the present invention.
Last aspect the present invention relates to polypeptide conjugate of the present invention is used for the purposes of multiple Industrial products, as is used for personal-care supplies and is used for cleaning composition.
The purpose of this invention is to provide polypeptide conjugate with the sex change originality that weakens.
As indicated above, well known in the art is by covalent attachment is connected one or several polymer molecule one or several polymer molecule is combined with a peptide carrier molecule with amino such as the peptide carrier molecule.Although confirmed already to be used to realize that the technology of this purpose can weaken the reaction of immunity system to polypeptide, there is some defective in this technology;
-because as the limited amount of the reached conjugated group (as amino) on many peptide molecules (being the common peptide molecule that molecular weight is about 10-200kDa) of Industrial products composition, the quantity of the polymer molecule that can put together with each peptide carrier molecule also is limited.
-because steric hindrance (being substrate to the low accessibility such as the avtive spot of the peptide molecule of enzyme), the activity (as catalytic activity) with polypeptide conjugate (as the enzyme conjugate) of a plurality of polymer molecules that are connected with each peptide carrier molecule is compared obvious reduction with corresponding parental generation peptide molecule.
The inventor has found that, can cause two or more peptide molecules and each polymer support molecule bonded conjugation techniques can weaken the sex change originality of the peptide molecule that is applicable to Industrial products.
And described polypeptide conjugate is also more stable than corresponding parental generation peptide molecule.
Compare with each peptide carrier molecule bonded polypeptide conjugate with several polymer molecules are arranged, polypeptide conjugate of the present invention also has the following advantages:
-the quantity of puting together the needed superfluous peptide molecule of process is less than accordingly requirement in the prior art that several polymer molecules and each peptide carrier molecule are puted together, because only need a conjugated group on the described peptide molecule.
-with prior art in have several polymer molecules to compare with each peptide carrier molecule bonded polypeptide conjugate, the activity of polypeptide conjugate of the present invention (as catalytic activity) is being kept to a greater extent.
Conjugate of the present invention
Therefore, first aspect, the polypeptide conjugate that the present invention relates to have the allergenicity that weakens, this conjugate comprise that has two or more polymeric carrier molecules of covalently bound peptide molecule with it.
Can described two or more peptide molecules be connected with described polymer support molecule with any currently known methods in this area.
In one embodiment of the present invention, described conjugate comprises that has one or several polymer support molecule of bonded peptide molecule with it, and above-mentioned intermolecular combination is that formed covalent linkage forms between one of two vinyl groups by divinylsulfone.
The total molecular weight of described polypeptide conjugate (Mr) scope is 50-40, and 000kDa is preferably 100-1000kDa, particularly 200-500kDa.
And conjugate of the present invention has 1-60, and preferred 2-40, an especially 3-20 peptide molecule combines with each polymer support molecule.
The peptide molecule that two or more are different is connected with each polymer support molecule and belongs to category of the present invention.Described not homopolypeptide can show different activities, as two kinds of different enzymic activitys.In addition, the function of described peptide molecule also can be different.
The example of the peptide molecule that function is different comprises enzyme molecule, ligand molecular, inhibitor molecules, acceptor molecule and antibody molecule.
In addition, can also provide to have with it for example enzyme molecule of bonded, as the conjugate of oxydase and effector molecule (being enhanser).Because the approximation of described two molecules can obtain a kind of highly effective conjugate.
The polymer support molecule
With the described polymer support molecule of described peptide molecule bonded can be any polymkeric substance with two above conjugated groups.Comprising natural or synthetic homopolymer, as polyvalent alcohol (promptly many-OH), polyamines is (promptly many-NH 2) and poly carboxylic acid (promptly many-as COOH), also to comprise heteropolymer, promptly have two different conjugated groups at least, as the polymkeric substance of hydroxyl and amido.
Described synthetic homopolymer and heteropolymer carrier molecule comprise star PEGs (Star-PEGs), branching PEGs (Branched PEGs), polyvinyl alcohol (PVA), polycarboxylate, poly--(V-Pyrol RC) and poly--D, L-amino acid.
Star PEGs is multiple-limb peg molecule (Gnanou etc., (1988), the Makromol.Chem.198.2885 that makes by the polymerization of the ethylene oxide molecule that comes from crosslinked Vinylstyrene core; Rein etc. (1993), Acta Polymer, 44,225).Star PEGs and branching PEGs all can be available from the Shearwater companies of the U.S..
The example of suitable natural homopolymer and heteropolymer has the dextran that comprises Sensor Chip CM 5, such as the Mierocrystalline cellulose of methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, Natvosol and hydroxypropylcellulose, the hydrolysate of chitosan, starch such as hydroxyethylamyle and hydroxypropylated starch, glycogen, agarose, guar gum, inulin, amylopectin, xanthene glue, carrageenin, pectin, alginic acid etc.
In one embodiment of the present invention, the molecular weight of described polymer support molecule is 1-10, and 000kDa is preferably 2-5,000kDa, especially 5-500kDa.
It is to be noted that all related among the present invention polymericular weights are molecular-weight average.
Peptide molecule
The peptide molecule that is connected with described polymer support molecule can be the peptide molecule of any kind.But, it is desirable to described peptide molecule certain type function is arranged.
Treat according to the inventive method in addition modified polypeptides can come from plant, animal or microorganism, but, preferably come from microbe-derived peptide molecule such as bacterium or fungi (promptly coming from filamentous fungus or yeast).
Special ideal peptide molecule is the albumen with antimicrobial, biology or enzymatic activity.
When described albumen was enzyme, this kind of enzyme can be selected from following one group: such as the lytic enzyme of proteolytic enzyme, lipase and cellulase, transferring enzyme, carbohydrase, and such as the oxydo-reductase of laccase and peroxidase, or phytase.
The molecular weight ranges that is used for most of enzyme of Industrial products is approximately 4-200kDa, preferred 15-150kDa, particularly 20-100kDa.
Part involved in the present invention molecular weight ranges in most of the cases is 100-2,000Da.
The inventor has found that the enzymatic activity of enzyme conjugate of the present invention is kept basically.
The activity that " substantially " kept in the context of the invention is meant with the activity of the peptide molecule of its parental generation unmodified and compares, its activity is at least 20-30%, is preferably 30-40%, more preferably 40-60%, be preferably 60-80%, more preferably 80-about 100%.
Method of the present invention
Second aspect the present invention relates to a kind of peptide molecule has the polypeptide conjugate of the allergenicity that weakens with acquisition method that is suitable for handling on a large scale.
According to the present invention, one or several polypeptide is connected on each polymer support molecule by following steps:
I) activate described polymer support molecule and
Ii) under the condition that is suitable for puting together, allow one or several peptide molecule and the molecular reaction of described activated polymer support and
Iii) seal the residual activity group on the described conjugate.
In step I) in can realize with any currently known methods in this area the activation of polymer support molecule.To introduce the example of these methods below.
The activation of polymer support molecule
The method of activating polypeptide molecule and conjugated polypeptide and chemistry were done fully to introduce in the literature.The method that is usually used in activating insoluble polymer molecule comprises with cyanogen bromide, Periodic acid, glutaraldehyde, di-epoxide, Epicholorohydrin, divinylsulfone, carbodiimide, sulfonic acid halide, three chlorotriazines etc. and activates functional groups (referring to R.F.Taylor, (1991), " protein immobilization.Function and application ", Marcel Dekker, N.Y.; S.S.Wong (1992), " protein is puted together and cross-linking chemistry ", CRC press, Boca Raton; G.T.Hermanson etc. (1993), " immobilization affinity ligand technology ", the academic press, N.Y.).Above-mentioned certain methods relates to activation insoluble polymer molecule, but also can be applicable to activate the soluble polymer molecule, for example, and Periodic acid, three chlorotriazines, sulfonic acid halide, divinylsulfone, carbodiimide etc.Functional group is amino, hydroxyl, thiol, carboxyl, aldehyde or the sulfydryl on the described polymer molecule, when selecting activation and puting together chemistry, must consider the conjugated group on the described peptide molecule of selection.
Also can use the method that the amino on activated polyvalent alcohol and the cytolysin is linked together by the electrophilic method.The leavings group that much is used for alcohol can produce the amine key.For example, can use alkylsulfonate such as tresylates (Nilsson etc. (1984), Enzymology method, the 104th volume, Jacoby, the W.B. work, academic press: Orlando, p.56-66; Nilsson etc. (1987), Enzymology method, the 135th volume; Mosbach, K., work; Academic press: Orlando, p.65-79; Scouten etc. (1987), Enzymology method, the 135th volume, Mosbach, K., work, academic press: Orlando, 1987; P.79-84; Crossland etc., (1971), J.Amr.Chem.Soc.1971,93, p.4217-4219), mesylate (Harris, (1985), JMS-REV.Macronol.Chem.Phys.C25,325-373; Harris etc. (1984), J.Polym.Sci.Polym.Chem. the 22nd edition, p.341-352), such as the aryl benzene sulfonate of tosylate and p-nitrophenyl sulfonate.
SULPHURYL CHLORIDE such as Tresyl chloride, hydroxyl on the multiple polymers molecule effectively can be converted into good leavings group (sulfonate), this group and can between polymer molecule and peptide molecule, form stable key during such as the reaction of the nucleophilic reagent of the amino on the polypeptide chain.Except height was puted together rate, reaction conditions generally was gentle (neutrality or weakly alkaline pH is to avoid sex change and seldom or not to destroy activity), and satisfied the nondestructive requirement of peptide molecule.
Can also produce the amine key with ethylene oxide group and other epoxide group.
To change into chloro-formic ester such as the polymer support molecule of polyvalent alcohol with carbonyl chloride, and can make the cytolysin base on the polypeptide chain produce amino-formate bond.This scheme can various deformation be implemented, and replaces chlorine with N-hydroxyl-succinimide, imidazoles, p-NP, DMAP.Described derivative is normally by allowing chloro-formic ester and the leavings group reaction that needs make.So these groups all can make described polypeptide produce amino-formate bond.
And, can produce urea and thiocarbamide with isocyanic ester and lsothiocyanates respectively.
Can obtain acid amides by polyhydroxy-acid with leavings group same as described above and ring-type imid throne.Can also use the poly-hydroxy succinate that obtains by reaction with succinyl oxide.Therefore it comprises and makes described conjugate be easier to the ester group of hydrolysis.This group can be activated by N-hydroxy-succinamide.
And, can introduce a kind of special joint.Using joint at most is cyanuryl chloride (Abuchowski etc., (1977), journal of biological chemistry, 252,3578-3581; Shafer etc., (1986), J.Polym.Sci.Polym.Chem. etc., the 24th edition, 375-378).
Be connected with the polymer support molecule of aromatic amine, carry out diazotization subsequently again, can produce a kind of very active diazonium salt, this salt can react with peptide molecule in position.The reaction that can also pass through the azlactone derivative of polyvalent alcohol produces amido linkage, thereby introduces another amido linkage.
The amino of peptide molecule can also be connected with polyvalent alcohol by amino-formate bond.Can be with lysine residue as skeleton.
In a kind of specific embodiments, two or more peptide molecules are connected on each polymer support molecule by following steps:
A) by connect a last active part activate described polymer support molecule and
B) allow two or more peptide molecules and the molecular reaction of described activated polymer support.
In one embodiment of the present invention, the activation in the step a) is to realize by an active part and its covalent attachment that will come from divinylsulfone.
For example in the WO 93/01498 that the Immunodex of Denmark K/S company is had, disclosed in detail and how to have used divinylsulfone, one or several peptide molecule has been conjugated on each polymer support molecule as conjugated group.
Composition
The invention still further relates to the composition that contains polypeptide conjugate of the present invention.
Described composition can also contain polypeptide, as albumen and/or enzyme and/or be usually used in composition in the following material: such as washing composition (comprising soap bar), household article, agrochemicals, personal-care supplies are as cleaning agent, for example, be used for the cleaning agent of latent eye glasses, makeup, cosmetics, oral and externally applied medicine is used to handle the composition of fabric, is used to clean the composition of crust, be used to produce the composition of food (as baking) and feed etc.
As the example of the polypeptide of enzyme comprise proteolytic enzyme, lipase, oxydo-reductase, carbohydrase, transferring enzyme, as trans-glutaminases, antimicrobial polypeptide and phytase.
Cleaning composition
Polypeptide conjugate of the present invention can be such as the laundry cleaning composition usually or wash a kind of composition that dishwashing is washed the cleaning composition of composition as the enzyme conjugate.Like this, the form of described enzyme in cleaning composition can be the form of dustless particle, stabilization liquid or protective enzyme.Dustless particle can be with such as being disclosed in US 4,106,991 and US 4,661,452 (being Novo Industri A/S has) in method production, and can optionally use the means known in the art dressing.The example of wax coating material have molecular-weight average be 1000-20000 poly-(oxyethane) product (polyoxyethylene glycol, PEG); Ethoxylized nonylphenol with 16-50 ethylene oxide unit(s); Ethoxylized fatty alcohol, alcohol wherein contain 12-20 carbon atom, and, 15-80 ethylene oxide unit(s) wherein arranged; Fatty Alcohol(C12-C14 and C12-C18); Lipid acid; Single, double and Witepsol W-S 55 with lipid acid.In the GB1483591 patent, disclosed the example of the film forming coating material that is applicable to fluidization.For instance, can be according to the method for having set up by adding polyvalent alcohol, sugar or sugar alcohol, lactic acid or boric acid stabilising liq zymin such as propylene glycol.Other enzyme stabilizers is known in the art.Can be according to being disclosed in EP238, the method in 216 prepares protective enzyme.
Cleaning composition of the present invention can be any common form, as pulvis, particle, paste or liquid.Liquid washing agent can be aqueous, contains usually up to 70% water and the organic solvent of 0-30%, or anhydrous.
Described cleaning composition contains one or several tensio-active agent, and wherein, each tensio-active agent can be anionic, non-ionic type, cationic or amphoteric ion type.Described washing composition contains the aniorfic surfactant of 0-50% usually, as linear alkylbenzene sulfonate (LAS), sulfonated (AOS), alkyl-sulphate (aliphatic alcohol sulfate) (AS), alcohol ethoxy vitriol (AEOS or AES), secondary sulfonated alkane (SAS), alpha-sulfo fatty acid methyl ester, alkyl or alkenyl succinic or soap.It can also contain the nonionic surface active agent of 0-40%, as alcohol ethoxylate (AEO or AE), pure propoxylated glycerine, carboxylated alcohol ethoxylate, nonyl phenol ethoxylate, alkyl poly glucoside, alkyl dimethyl amine oxide, ethoxylated fatty acid single ethanol amide or polyhydroxy alkyl fatty acid amide (for example, disclosed in the WO 92/06154).
Described cleaning composition can also contain one or more enzymes, as amylase, Starch debranching enzyme, esterase, lipase, at, proteolytic enzyme, cellulase, peroxidase or oxydase, for example, laccase, and antimicrobial polypeptide.Can be according to the present invention to such as a kind of, several of the described polypeptide of enzyme or all modify (promptly puting together).
Usually, described washing composition contains the washing auxiliary detergent of 1-65%, but some is washed the dish washing composition even can contain washing auxiliary detergent up to 90%, or coordination agent, as zeolite, diphosphate, triphosphate, phosphonate, Citrate trianion, nitrilotriacetic acid(NTA) (NTA), ethylenediamine tetraacetic acid (EDTA) (EDTA), diethylene triaminepentaacetic acid(DTPA) (DTMPA), alkyl-or alkenyl succinic, soluble silicate or layered silicate (for example, available from Hoechst SKS-6).
Described washing auxiliary detergent can be divided into phosphorous type and without phosphorus type.The example of inorganic phosphor-contained alkaline washing auxiliary agent comprises water-soluble salt, particularly alkali metal pyrophosphate, orthophosphoric acid salt, polyphosphate and phosphonate.The example of without phosphorus type inorganic assistant agent comprises water soluble alkali metal carbonate, borate and silicate, and stratiform bisilicate and crystallization of all kinds water-insoluble or amorphous silicon aluminate, and wherein, known foremost representative is a zeolite.
The example of suitable organic additive comprises an alkali metal salt, ammonium salt or the substituted ammonium salt of succsinic acid, propanedioic acid, lipid acid propanedioic acid, lipid acid sulfonic acid, carboxy methoxy-succinic acid, poly-acetate, carboxylic acid, poly carboxylic acid, aminopolycarboxylic and poly-acetyl carboxylic acid.
Described washing composition can also be not composite,, does not have washing auxiliary detergent basically that is.
Described washing composition can comprise one or more polymkeric substance.Its example has carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyoxyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylate such as polyacrylic ester, polymaleic acid ester, toxilic acid/acrylic copolymer and methylacrylic acid Lauryl Ester/acrylic copolymer.
Described detergent composition can contain chlorine/bromine type or aerobic type SYNTHETIC OPTICAL WHITNER.Described SYNTHETIC OPTICAL WHITNER can be capsule type coating type or non-.The example of inorganic chlorine/bromine type SYNTHETIC OPTICAL WHITNER has hypochlorous acid or hypobromous acid lithium, sodium or calcium, or Efficacious Disinfeitant.This bleaching system can also contain H 2O 2The source, as perborate or percarbonate, it can make up with the peracid bleach activator that becomes such as tetrem acyl ethylene diamine (TAED) or nonanoly acyloxy benzene sulfonate (NOBS).
The example of organochlorine/bromine type SYNTHETIC OPTICAL WHITNER has heterocycle N-bromine and N-chlorine imide, as TCCA (Trichloroisocyanuric acid), tribromo isocyanuric acid, dibromo isocyanuric acid and dichloroisocyanuric acid, and with the salt of water-soluble cationic such as potassium and sodium.Hydantoin compound also is suitable for.Described bleaching system can also comprise peroxy acid, as acid amides, imide and sulfone type.
In washing the dish washing composition, the preferred described oxygen bleaching agent that has as the SYNTHETIC OPTICAL WHITNER of inorganic peracid salt form, preferably uses bleach precursor or peracetic acid compound.The example of common suitable peroxy bleaching compound has the tetrahydrate and the monohydrate of alkali metal perborate, alkali metal percarbonate, persilicate and superphosphate.Preferred activator material has TAED or NOBS.
The enzyme of cleaning composition of the present invention can be stable with conventional stablizer, for example, polyvalent alcohol such as propylene glycol or glycerine, sugar or sugar alcohol, lactic acid, boric acid, or boric acid derivatives are as aromatic borate, and described composition can be according to preparing such as the method that is disclosed among WO 92/19709 and the WO92/19708.Enzyme of the present invention can also pass through to add the reversible enzyme inhibitors, as the protein inhibitor that is disclosed among the EP 0544777B1 is stablized.
Described washing composition can also contain other conventional detergent ingredients, as fabric regulator, comprise clay, deflocculate material, profoamer/froth suppressor (in washing the dish washing composition, being froth suppressor), suds suppressor, sanitas, soil-suspending agent, anti-soil dirt deposition agent, dyestuff, dewatering agent, sterilant, white dyes or spices again.
It is neutral or alkaline that its pH (measuring in the aqueous solution under working concentration) is generally, for example in the 7-11 scope.
The specific form of the laundry cleaning composition in the scope of the invention comprises:
1) make the particulate cleaning composition that tap density is at least 600g/l, it contains:
Linear alkylbenzene sulfonate (in acid) 7-12％
Alcohol ethoxy vitriol (for example, C 12-18Alcohol, 1-2EO) or alkyl-sulphate (C for example 16-18) 1-4％
Alcohol ethoxylate (for example, C 14-15Alcohol, 7EO) 5-9％
Yellow soda ash is (as Na 2CO 3) 14-20％
Soluble silicate is (as Na 2O，2SiO 2) 2-6％
Zeolite is (as NaAlSiO 4) 15-22％
Sodium sulfate is (as Na 2SO 4) 0-6％
Trisodium Citrate/citric acid is (as C 6H 5Na 3O 7/C 6H 8O 7) 0-15％
Sodium peroxoborate is (as NaBO 3.H 2O) 11-18％
TAED 2-6％
Carboxymethyl cellulose 0-2％
Polymkeric substance (as toxilic acid/acrylic copolymer, PVP, PEG) 0-3％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, suds suppressor, spices, white dyes, optical bleaching agent) 0-5％
2) make the particulate cleaning composition that tap density is at least 600g/l, it contains
Linear alkylbenzene sulfonate (in acid) 6-11％
Alcohol ethoxy vitriol (for example, C 12-18Alcohol, 1-2 EO) or alkyl-sulphate (for example, C 16-18) 1-3％
Alcohol ethoxylate (as, C 14-15Alcohol, 7EO) 5-9％
Yellow soda ash is (as Na 2CO 3) 15-21％
Soluble silicate is (as Na 2O，2SiO 2) 1-4％
Zeolite is (as NaAlSiO 4) 24-34％
Sodium sulfate is (as Na 2SO 4) 4-10％
Trisodium Citrate/citric acid is (as C 6H 5Na 3O 7/C 6H 8O 7) 0-15％
Carboxymethyl cellulose 0-2％
Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG) 1-6％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, suds suppressor, spices) 0-5％
3) make the particulate cleaning composition that tap density is at least 600g/l, it contains
Linear alkylbenzene sulfonate (in acid) 5-9％
Alcohol ethoxylate (for example, C 12-15Alcohol, 7EO) 7-14％
Soap is as lipid acid (for example, C 16-22, lipid acid) 1-3％
Yellow soda ash is (as Na 2CO 3) 10-17％
Soluble silicate is (as Na 2O，2SiO 2) 3-9％
Zeolite is (as NaAlSiO 4) 23-33％
Sodium sulfate is (as Na 2SO 4) 0-4％
Sodium peroxoborate is (as NaBO 3.H 2O) 8-16％
TAED 2-8％
Phosphonate (as EDTMPA) 0-1％
Carboxymethyl cellulose 0-2％
Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG) 0-3％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, suds suppressor, spices, white dyes) 0-5％
4) make the particulate cleaning composition that tap density is at least 600g/l, it contains
Linear alkylbenzene sulfonate (in acid) 8-12％
Alcohol ethoxylate (for example, C 12-15Alcohol, 7EO) 10-25％
Yellow soda ash is (as Na 2CO 3) 14-22％
Soluble silicate is (as Na 2O，2SiO 2) 1-5％
Zeolite is (as NaAlSiO 4) 25-35％
Sodium sulfate is (as Na 2SO 4) 0-10％
Carboxymethyl cellulose 0-2％
Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG) 1-3％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, suds suppressor, spices) 0-5％
5) aqueous liquid detergent composition, it contains
Linear alkylbenzene sulfonate (in acid) 15-21％
Alcohol ethoxylate (for example, C 12-15Alcohol, 7EO or C 12-15Alcohol, 5EO) 12-18％
Soap is as lipid acid (for example, oleic acid) 3-13％
Alkenyl succinic (C 12-14) 0-13％
Monoethanolamine 8-18％
Citric acid 2-8％
Phosphonate 0-3％
Polymkeric substance (for example, PVP, PEG) 0-3％
Borate is (as B 4O 7) 0-2％
Ethanol 0-3％
Propylene glycol 8-14％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, dispersion agent, suds suppressor, spices, white dyes) 0-5％
6) liquid detergent compositions of water structure, it contains
Linear alkylbenzene sulfonate (in acid) 15-21％
Alcohol ethoxylate (for example, C 12-15Alcohol, 7EO, or C 12-15Alcohol, 5EO) 3-9％
Soap is as lipid acid (for example, oleic acid) 3-10％
Zeolite is (as NaAlSiO 4) 14-22％
Tripotassium Citrate 9-18％
Borate is (as B 4O 7) 0-2％
Carboxymethyl cellulose 0-2％
Polymkeric substance (for example, PEG, PVP) 0-3％
Conjugated polymer is as methylacrylic acid dodecyl ester/acrylic copolymer; Mol ratio 25: 1; MW 3800 0-3％
Glycerine 0-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, dispersion agent, suds suppressor, spices, white dyes) 0-5％
7) make the particulate cleaning composition that tap density is at least 600g/l, it contains
Aliphatic alcohol sulfate 5-10％
The ethoxylated fatty acid single ethanol amide 3-9％
Soap is as lipid acid 0-3％
Yellow soda ash is (as Na 2CO 3) 5-10％
Soluble silicate is (as Na 2O，2SiO 2) 1-4％
Zeolite is (as NaAlSiO 4) 20-40％
Sodium sulfate is (as Na 2SO 4) 2-8％
Sodium peroxoborate is (as NaBO 3.H 2O) 12-18％
TAED 2-7％
Polymkeric substance (as, toxilic acid/acrylic copolymer, PEG) 1-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, white dyes, suds suppressor, spices) 0-5％
8) make the particulate cleaning composition, it contains
Linear alkylbenzene sulfonate (in acid) 8-14％
The ethoxylated fatty acid single ethanol amide 5-11％
Soap is as lipid acid 0-3％
Yellow soda ash is (as Na 2CO 3) 4-10％
Soluble silicate is (as Na 2O，2SiO 2) 1-4％
Zeolite is (as NaAlSiO 4) 30-50％
Sodium sulfate is (as Na 2SO 4) 3-11％
Trisodium Citrate is (as C 6H 5Na 3O 7) 5-12％
Polymkeric substance is (as PVP, toxilic acid/acrylic copolymer, PEG) 1-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, suds suppressor, spices) 0-5％
9) make the particulate cleaning composition, it contains
Linear alkylbenzene sulfonate (in acid) 6-12％
Nonionic surface active agent 1-4％
Soap is as lipid acid 2-6％
Yellow soda ash is (as Na 2CO 3) 14-22％
Zeolite is (as NaAlSiO 4) 18-32％
Sodium sulfate is (as Na 2SO 4) 5-20％
Trisodium Citrate is (as C 6H 5Na 3O 7) 3-8％
Sodium peroxoborate is (as NaBO 3.H 2O) 4-9％
Bleach activator (as NOBS or TAED) 1-5％
Carboxymethyl cellulose 0-2％
Polymkeric substance (for example, polycarboxylate or PEG) 1-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, white dyes, spices) 0-5％
10) aqueous liquid detergent composition
Linear alkylbenzene sulfonate (in acid) 15-23％
Alcohol ethoxy vitriol (for example, C 12-15Alcohol, 2-3EO) 8-15％
Alcohol ethoxylate (for example, C 12-15Alcohol, 7EO, or C 12-15Alcohol, 5EO) 3-9％
Soap is as lipid acid (for example, lauric acid) 0-3％
Monoethanolamine 1-5％
Trisodium Citrate 5-10％
Hydrotropic agent (as toluenesulfonic acid sodium salt) 2-6％
Borate is (as B 4O 7) 0-2％
Carboxymethyl cellulose 0-1％
Ethanol 1-3％
Propylene glycol 2-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, polymkeric substance, spices, white dyes) 0-5％
11) aqueous liquid detergent composition, it contains
Linear alkylbenzene sulfonate (in acid) 20-32％
Alcohol ethoxylate (for example, C 12-15Alcohol, 7EO, or C 12-15Alcohol, 5EO) 6-12％
Monoethanolamine 2-6％
Citric acid 8-14％
Borate is (with B 4O 7Meter) 1-3％
Polymkeric substance (for example toxilic acid/acrylic copolymer, conjugated polymer is as methylacrylic acid Lauryl Ester/acrylic copolymer) 0-3％
Glycerine 3-8％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, hydrotropic agent, dispersion agent, spices, white dyes) 0-5％
12) make the particulate cleaning composition that tap density is at least 600g/l, it contains
Aniorfic surfactant (linear alkylbenzene sulfonate, alkyl-sulphate, sulfonated, alpha-sulfo fatty acid methyl ester, sulfonated alkane, soap) 25-40％
Nonionic surface active agent (for example, alcohol ethoxylate) 1-10％
Yellow soda ash is (as Na 2CO 3) 8-25％
Soluble silicate is (as Na 2O，2SiO 2) 5-15％
Sodium sulfate is (as Na 2SO 4) 0-5％
Zeolite is (as NaAlSiO 4) 15-28％
Sodium peroxoborate is (as NaBO 3.4H 2O) 0-20％
Bleach activator (TAED or NOBS) 0-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (as spices, white dyes) 0-3％
13) as above-mentioned 1)-12) detergent compositions, wherein, all or part of linear alkylbenzene sulfonate is by (C 12-C 18) alkyl-sulphate substitutes.
14) make the particulate cleaning composition that tap density is at least 600g/l, it contains
(C 12-C 18) alkyl-sulphate 9-15％
Alcohol ethoxylate 3-6％
The polyhydroxy alkyl fatty acid amide 1-5％
Zeolite is (as NaAlSiO 4) 10-20％
Stratiform bisilicate (for example, available from Hoechst SK56) 10-20％
Yellow soda ash (Na 2CO 3) 3-12％
Soluble silicate is (as Na 2O，2SiO 2) 0-6％
Trisodium Citrate 4-8％
SPC-D 13-22％
TAED 3-8％
Polymkeric substance (as, polycarboxylate and PVP) 0-5％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (for example, white dyes, optical bleaching agent, spices, suds suppressor) 0-5％
15) make the particulate cleaning composition that tap density is at least 600g/l, it contains
(C 12-C 18) alkyl-sulphate 4-8％
Alcohol ethoxylate 11-15％
Soap 1-4％
Zeolite MAP or zeolite A 35-45％
Yellow soda ash (Na 2CO 3) 2-8％
Soluble silicate is (as Na 2O，2SiO 2) 0-4％
SPC-D 13-22％
TAED 1-8％
Carboxymethyl cellulose 0-3％
Polymkeric substance (as, polycarboxylate and PVP) 0-3％
The enzyme (in pure enzyme protein) that comprises modifying enzyme 0.0001-0.5％
Trace ingredients (as white dyes, phosphonate, spices) 0-3％
16) as 1)-15) described in detergent compositions, it contains peracid a kind of stabilization or encapsulate, perhaps as a kind of extra composition or as the surrogate of the bleaching system of having illustrated.
17) as 1), 3), 7), 9) and 12) described in cleaning composition, wherein, perborate is substituted by percarbonate.
18) as 1), 3), 7), 9), 12), 14) and 15) described in cleaning composition, it also additionally contains a kind of Mn catalyst.For example, this Mn catalyst can be " the effective Mn catalyst that is used for cold bleaching " literary composition one of the compound that reveals of slope (nature, 369, (1994), p.637-639).
19) make the cleaning composition of anhydrous wash liquid composition, it contains liquid nonionic surfactants, as straight chain alkoxylate primary alconol, adjuvant systems (for example, phosphoric acid salt), enzyme and alkali.Described washing composition can also contain aniorfic surfactant and/or bleaching system.
The dishwashing of washing in the scope of the invention is washed the specific form of composition and is comprised:
1) powdery is washed the dish composition automatically
Nonionic surface active agent 0.4-2.5％
Water glass 0-20％
Sodium disilicate 3-20％
Tri sodium Phosphate 20-40％
Yellow soda ash 0-20％
Sodium peroxoborate 2-9％
Tetrem acyl ethylene diamine (TAED) 1-4％
Sodium sulfate 5-33％
The enzyme that comprises modifying enzyme 0.0001-0.5％
2) powdery is washed the dish composition automatically
Nonionic surface active agent (for example, alcohol ethoxylate) 1-2％
Sodium disilicate 2-30％
Yellow soda ash 10-50％
Alendronate 0-5％
Citrate trisodium dihydrate 9-30％
Sodium nitrilo triacetate (NTA) 0-20％
The Sodium peroxoborate monohydrate 5-10％
Tetrem acyl ethylene diamine (TAED) 1-2％
Acrylic acid polymer (for example, toxilic acid/acrylic copolymer) 6-25％
The enzyme that comprises modifying enzyme 0.0001-0.5％
Spices 0.1-0.5％
Water 5-10％
3) powdery is washed the dish composition automatically
Nonionic surface active agent 0.5-2.0％
Sodium disilicate 25-40％
Trisodium Citrate 30-55％
Yellow soda ash 0-29％
Sodium bicarbonate 0-20％
The Sodium peroxoborate monohydrate 0-15％
Tetrem acyl ethylene diamine (TAED) 0-6％
Toxilic acid/acrylic copolymer 0-5％
Clay 1-3％
Poly-(amino acid) 0-20％
Sodium polyacrylate 0-8％
The enzyme that comprises modifying enzyme 0.0001-0.5％
4) powdery is washed the dish composition automatically
Nonionic surface active agent 1-2％
Zeolite MAP 15-42％
Sodium disilicate 30-34％
Trisodium Citrate 0-12％
Yellow soda ash 0-20％
The Sodium peroxoborate monohydrate 7-15％
Tetrem acyl ethylene diamine (TAED) 0-3％
Polymkeric substance 0-4％
Toxilic acid/acrylic copolymer 0-5％
Organic phosphonate 0-4％
Clay 1-2％
The enzyme that comprises modifying enzyme 0.0001-0.5％
Sodium sulfate Surplus
5) powdery is washed the dish composition automatically
Nonionic surface active agent 1-7％
Sodium disilicate 18-30％
Trisodium citrate 10-24％
Yellow soda ash 12-20％
Single persulphate (2KHSO 5·KHSO 4·K 2SO 4) 15-21％
Bleaching stibilizer 0.1-2％
Toxilic acid/acrylic copolymer 0-6％
Diethylene triaminepentaacetic acid(DTPA), five sodium-salt 0-2.5％
The enzyme that comprises modifying enzyme 0.0001-0.5％
Sodium sulfate, water Surplus
6) powdery and the liquid with cleansing surfactants system is washed the dish composition
Nonionic surface active agent 0-1.5％
Octadecyl dimethyl amine N-oxide dihydrate 0-5％
80 of octadecyl dimethyl amine N-oxide dihydrate and hexadecyldimethyl benzyl ammonium amine n-oxide dihydrate: 20wt.C18/C16 mixture 0-4％
70 of anhydrous octadecyl two (hydroxyethyl) amine n-oxide and anhydrous hexadecyl two (hydroxyethyl) amine n-oxide: 30wt.C18/C16 mixture 0-5％
Average degree of ethoxylation is 3 C 13-C 15Alkyl ethoxy sulfate 0-10％
Average degree of ethoxylation is 3 C 12-C 15Alkyl ethoxy sulfate 0-5％
Average degree of ethoxylation is 12 C 13-C 15Ethoxylated alcohol 0-5％
Average degree of ethoxylation is 9 C 12-C 15Ethoxylated alcohol mixture 0-6.5％
Average degree of ethoxylation is 30 C 13-C 15Ethoxylated alcohol mixture 0-4％
Sodium disilicate 0-33％
Tri sodium Phosphate 0-46％
Trisodium Citrate 0-28％
Citric acid 0-29％
Yellow soda ash 0-20％
The Sodium peroxoborate monohydrate 0-11.5％
Tetrem acyl ethylene diamine (TAED) 0-4％
Toxilic acid/acrylic copolymer 0-7.5％
Sodium sulfate 0-12.5％
The enzyme that comprises modifying enzyme 0.0001-0.5％
7) anhydrous liq is washed the dish composition automatically
Liquid nonionic type tensio-active agent (for example, alcohol ethoxylate) 2.0-10.0％
Alkalimetal silicate 3.0-15.0％
Alkali metal phosphate 20.0-40.0％
Be selected from the liquid vehicle of higher glycols, polyoxyethylene glycol, polyoxide and glycol ether 25.0-45.0％
Stablizer (for example, phosphoric acid and C 16-C 18The partial ester of alkanol) 0.5-7.0％
Froth suppressor (for example, siloxanes) 0-1.5％
The enzyme that comprises modifying enzyme 0.0001-0.5％
8) anhydrous liq is washed the dish composition
Liquid nonionic surfactants (for example, alcohol ethoxylate) 2.0-10.0％
Water glass 3.0-15.0％
Alkaline carbonate 7.0-20.0％
Trisodium Citrate 0.0-1.5％
Stable system (as the mixture of siloxanes in small, broken bits and lower molecular weight dialkyl group polyglycol ether) 0.5-7.0％
The low-molecular-weight polypropylene acid polymer 5.0-15.0％
The clay gel thickening material (as, wilkinite) 0.0-10.0％
Hydroxypropyl cellulose polymer 0.0-0.6％
The enzyme that comprises modifying enzyme 0.0001-0.05％
Be selected from the liquid vehicle of higher glycols, polyoxyethylene glycol, polyoxide and glycol ether Surplus
9) thixotropic liquid is washed the dish composition automatically
C 12-C 14Lipid acid 0-0.5％
Block copolymer surfactant 1.5-15.0％
Trisodium Citrate 0-12％
Tri sodium Phosphate 0-15％
Yellow soda ash 0-8％
Aluminium Tristearate Micronized sterile 0-0.1％
Cumene sodium sulfonate 0-1.7％
Polyacrylate thickeners 1.32-2.5％
Sodium polyacrylate 2.4-6.0％
Boric acid 0-4.0％
Sodium formiate 0-0.45％
Calcium formiate 0-0.2％
Oxidation n-decyl phenylbenzene sodium disulfonate 0-4.0％
Monoethanolamine (MEA) 0-1.86％
Sodium hydroxide (50%) 1.9-9.3％
1, the 2-propylene glycol 0-9.4％
The enzyme that comprises modifying enzyme 0.0001-0.5％
Suds suppressor, dyestuff, spices, water Surplus
10) liquid is washed the dish composition automatically
Alcohol ethoxylate 0-20％
The fatty acid ester sulphonate 0-30％
Sodium lauryl sulphate 0-20％
Alkyl poly glucoside 0-21％
Oleic acid 0-10％
The sodium disilicate monohydrate 18-33％
Trisodium citrate dihydrate 18-33％
Sodium stearate 0-2.5％
The Sodium peroxoborate monohydrate 0-13％
Tetrem acyl ethylene diamine (TAED) 0-8％
Toxilic acid/acrylic copolymer 4-8％
The enzyme that comprises modifying enzyme 0.0001-0.5％
11) contain the liquid of protecting bleaching particle and wash the dish composition automatically
Water glass 5-10％
Tetrapotassium pyrophosphate 15-25％
Tri sodium Phosphate 0-2％
Salt of wormwood 4-8％
The protection bleaching particle is as chlorine 5-10％
Polymeric viscosifier 0.7-1.5％
Potassium hydroxide 0-2％
The enzyme that comprises modifying enzyme 0.0001-0.5％
Water Surplus
11) as 1), 2), 3), 4), 6) and 10) described in wash the dish composition automatically, wherein, perborate is substituted by percarbonate.
12) as 1)-6) the described dish composition of washing automatically, it additionally contains a kind of Mn catalyst.For example, this Mn catalyst can be one of compound disclosed in " the effective Mn catalyst that is used for cold bleaching " literary composition (nature, 369, (1994), p.637-639).
Personal care applications
Enzyme conjugate with the allergenicity that weakens of the present invention also can be used for personal-care supplies.
Proteolytic enzyme
Proteolytic enzyme is the well-known proteolytic enzyme that is used for cleaning contact lenses.Its protein dirt on can the hydrolysis eyeglass, and therefore with its dissolving.For using comfort, the removing of albumen dirt is necessary.
Proteolytic enzyme still is the effective constituent of skin clean goods, and it can remove dead Keratin sulfate skins cell, thereby makes skin look whiter, tenderer.
Also proteolytic enzyme can be used for oral care implement,, but also can be used in the tooth powder in particular for the cleaning artificial tooth.
And proteolytic enzyme can be used for comprising shampoo, conditioning agent, washing lotion, emulsifiable paste, soap bar, cosmetic perfumed soap and liquid soap in beauty treatment, bathing and the shower goods.
Lipase
Lipase can be used for makeup as the activeconstituents of skin clean goods and anti-acne goods, removing unnecessary sebum, and be used for such as the bathing of emulsifiable paste and washing lotion and shower goods being used for skin care as activeconstituents.
The goods (for example shampoo) that also lipase can be used for having one's hair wash are with sebum and other lipid matter of effective removing hair surface.
Lipase still is used to clean the effective constituent of the goods of contact lens, and it can remove the lipid deposits of lens surface.
Oxydo-reductase
The modal oxydo-reductase that is used for the personal care purpose is oxydase (often being notatin), has and can guarantee to produce H 2O 2Substrate (as glucose), then will be such as SCN by peroxidase (often being lactoperoxidase) -Or I -Be oxidized to biocide (SCNO -Or I 2).This enzyme complex itself is known, as comes from breast and saliva.
The antimicrobial system that it is used as mouth care goods (collutory, tooth powder, chewing gum) is commercial applications in addition, wherein, also it can be mixed with amyloglucosidase, to produce glucose.Known this system also can be used for being used for anticorrosion purpose in the cosmetic product.
The known antimicrobial system that contains the combination of a kind of oxydase and a kind of peroxidase can be used for cleaning contact lenses.
The another kind of purposes of oxydo-reductase is to carry out oxidative hair dyeing with oxydase, peroxidase and laccase.
The free radical that is generated on known skin (with the hair) surface is relevant with the weathering process (hair degenerates) of skin.
Described free radical can activate the chain reaction that can cause panniculus, collagen and cytoclasis.To be used for makeup such as the free-radical scavengers of superoxide-dismutase and be well-known (R.L.Goldemberg, DCI, Nov.93, p.48-52).
Protein disulfide isomerase (PDI) also is a kind of oxydo-reductase.Can use it for hair-waving (waving of hair) (with the disulfide bond reduction in the hair and reoxidize) and repair impaired hair (wherein, described damage mainly be reduction existing disulfide linkage).
Carbohydrase
Formed dental plaque mainly is made up of polysaccharide on dental surface.These polysaccharide adhere to tooth and microorganism surface.Described polysaccharide mainly is α-1,6 in conjunction with glucose (dextran) and α-1,3 in conjunction with glucose (mutan).Help the adhesive matrix of hydrolysis dental plaque such as the dissimilar dextranase of MUTANASE and dextranase, make it be easy to remove by mechanical effect.
Under the effect of dextranase, can also remove the microbial film that is created on such as biomembranous other type on the contact lens.
Antimicrobial polypeptide
Antimicrobial polypeptide has purposes widely, as is used for the anticorrosion of makeup, anti-acne goods, reodorant and shampoo.This class polypeptide also can be used for the contact lens goods.
Concrete formula for a product
The example of concrete personal care composition is disclosed in " makeup and cosmetics " (WilfriedUmbach work, Ellis Horwood company limited publishes, England (1991)) and " tensio-active agents in the consuming product " (J.Falbe work, Spring-Verlag publishes (1987)).
Given below is that non-exhaustive instructs prescription.These prescriptions have constituted the general view of the important personal care product prescription that the present invention relates to.
Perfumed soap
Composition Example ％
Surfactant chelating agent consistency modifiers dye fluorescence brightening agent antioxidant brightening agent aromatic enzyme water Soap (sodium salt) ethylenediamine tetra-acetic acid NaCl 2; Two (1,1-dimethyl ethyl)-4-methylphenol (BHT) titanium white protease/lipase of 6- About 0.5＜0.1＜0.1 0.1-0.3 0.1-0.3 1.0-2.0 0-5 surplus of 83-87 0.1-0.3
Synthetic detergent
Composition Example ％
Surfactant re-esterified agent plasticizer filler activating agent dyestuff aromatic enzyme water Lauryl sulfate dodecyl sulfosuccinate fatty alcohol stearoyl list/double glyceride starch salicylic acid protease/lipase 30-50 1-12 10-20 0-10 0-10 0-1＜0.2 0-2 0-5 surplus
Foam washing lotion and shower washing lotion
Composition Example % foam washing lotion % shower washing lotion
Tensio-active agent is fat agent enzyme again Lauryl ether sulfate cocoa aminopropyl dimethyl betaine ethoxylated fatty acid Fatty Alcohol(C12-C14 and C12-C18) ethoxylized fatty alcohol proteolytic enzyme/lipase 10-20 2-4 0.5-2 0.5-3 0.5-5 0-5 10-12 2-4 - 0-4 0-5
Composition Example % foam washing lotion % shower washing lotion
Foam stabiliser conditioning agent thickener pearling agent activating agent preservatives, dyes aromatic enzyme water The quaternized hydroxypropyl cellulose NaCl of Marlamid glycol stearate plant extracts 5-bromo-5-nitro-1,3-diox protease/lipase 0.2-2-0-3 0-2 0-1 0.1 0.1-0.2 0.3-3 0-5 surplus 0-4 0-0.5 0-3-0-1 0.1 0.1 0.3-2 0-5 surpluses
Protective skin cream (water-in-oil-type and oil-in-water-type)
Composition Example The % water-in-oil-type / oil-in-water-type
The emulsifying agent fat derivant Sesquialter oleic acid Isosorbide Dinitrate aluminum stearate triethanolamine stearate cetyl/octadecanoyl alcohol polyglycol ether isopropyl palmitate cetyl/octadecanoyl alcohol 3-5 1-2 - - 1-5 - - - 1-2 1-3 0-3 0-2
Wetting agent stabilizing agent anticorrisive agent enzyme water 2-octyldodecanol stearic/palmitic acids caprylic/capric triglyceride tristerin glycerine sorbierite many (hydroxycarboxylic acid) propane diols magnesium sulfate p-hydroxybenzoate protease/lipase 2-10-5-10-1-5 1-5 0.5-2-0-0.8 0.2-0.4 0-5 surplus 3-7 0-3-0-5 1-5 1-5-0-3-0.2-0.4 0-5 surplus
The clean body washing lotion (oil-in-water-type) and the skin care washing lotion that are used for moist skin
Composition Example The clean body washing lotion of % % skin care washing lotion
Emulsifying agent fat derivant wetting agent thickener anticorrisive agent enzyme Cetyl/octadecanoyl alcohol polyglycol ether mono laurate Isosorbide Dinitrate odium stearate Sodium Lauryl Ether Sulphate 2-octyldodecanol paraffin oil beeswax Ethylhexyl stearate isopropyl palmitate glycerine sorbierite polyacrylate methylhydroxypropylcellulose p-hydroxybenzoate protease/lipase 1-3 0.5-1 - - 1-3 - 0.5-1 3-7 - 3-5 - 0-0.3 0-0.3 0.2-0.4 0-5 - - 1-2 0.5-2 0-5 20-25 - - 2-5 5-10 0-5 0-1 0-0.5 0.2-0.4 0-5
Water Surplus Surplus
Face washing liquid
Composition Example ％
Surfactant re-esterified agent solubilizer cleaning and renewal composition wetting agent anticorrisive agent astringent counter-stimulus enzyme water Lauryl ether magnesium sulfate Di-n-butyl Adipate castor oil polyglycol ether ethanol glycerine sorbierite p-hydroxybenzoate plant extracts panthenol allantoin plant extracts protease/lipase 0.2-0.5 1-2 0.1-1 0-15 0-5 0-5 0.2-0.4 1-5 0-1 0-0.2 0.5-3 0-5 surplus
Shampoo
Composition Example ％
Surfactant foam booster conditioning agent re-esterified agent additive anticorrisive agent pearling agent Lauryl ether sulfate coconut fatty acid aminopropyl dimethyl betaine fatty acid polyethylene glycol ester fatty acid ethanol amide quaternized hydroxyethylcellulos protein hydrolysate ethoxylation lanolin alcohol anti-dandruff agent 5-bromo-5-nitro-1,3-diox glycol stearate 12-16 2-5 0-2 0.5-2.5 0.4-1 0.2-1 0.2-1 0-1 0.1-0.3 0-2
Dyestuff pH regulator agent perfume compound enzyme water Acid/basonuclin enzyme/lipase ＜0.1 0.1-1 0.3-0.5 0-5 surplus
Hair moistening liquid (hair rinse) and hair regulator solution
Composition Example ％ ％
Surfactant re-esterified agent consistency modifiers thickener conditioning agent preservatives, dyes pH adjusting agent aromatic enzyme water Fatty alcohol polyglycol ether hexadecyltrimethylammonium chloride dimethyl benzyl stearyl ammonium chloride cetyl/octadecanoyl list/two glyceride fatty alcohol methylhydroxypropylcellulose quaternized hydroxyethylcellulos p-hydroxybenzoate acid/basonuclin enzyme/lipase Hair moistening liquid 0.1-0.2 0.5-1-0.5-1.5 1-2.5 0.3-0.6 0.1-0.3 0.1-0.3＜0.1 0.1-1 0.2-0.5 0-5 surplus Hair regulator solution 1.5-2.5-0.5-1 1.5-2.5 2.5-3.5 0.4-0.8 0.3-0.4 0.1-0.3＜0.1 0.1-1 0.2-0.5 0-5 surplus
Shampoo
Composition Example ％
Component 1: tensio-active agent consistency modifiers Basic dyeing cream lauryl ether sulfate ethoxylated castor oil Fatty Alcohol(C12-C14 and C12-C18) 1-4 1-2 8-10
Reducing agent buffer solution chelating agent alkaline agent oxidation dye enzyme water component II: surfactant oxidant stabilizing agent thickener enzyme water Sodium sulfite ammonium chloride 1-hydroxyl ethane-1; 1-di 2 ethylhexyl phosphonic acid ammonia developer coupling agent laccase hydrogen peroxide dispersion lauryl ether sulfate hydrogen peroxide 1-hydroxyl ethane-1,1-di 2 ethylhexyl phosphonic acid polyacrylate laccase 0.8-1.2 0.5-1 0.1-0.2 1.2-2 11 0-5 surplus 0.5-1 6-9 1-1.5 3-5 0-5 surpluses
Shaving cream
Composition Example ％
Soap fat constituent stabilizing agent enzyme water Palmitic acid/stearic acid potassium hydroxide NaOH coconut oil polyethylene glycol sodium tetraborate sodium metasilicate sorbierite protease 30-40 5-7 1-2 5-10 0-2 0-0.5 0-0.5 0-3 0-5 surplus
Liquid shaves
Composition Example ％
Sterilization and phenolic acid (phonic acid) re-esterified agent solubilizer astringent counter-stimulus stabilizing agent enzyme water Ethanol Di-n-butyl Adipate ethoxylated castor oil plant extracts panthenol plant extracts glycerine sorbierite propane diols protease 40-80 1-2 0.5-1 1-10 0-0.5 0-2 0-5 0-5 0-3 0-5 surplus
The hair washing face cream
Composition Example ％
The agent of consistency modifiers vaseline antioxidant aromatic dyestuff enzyme bating Fatty alcohol ethoxylation lanolin alcohol vaseline branched paraffins 2; Two (1,1-dimethyl ethyl)-4-methylphenol (BHT) lipase glycerine of 6- 4-5 3-6 45-52 10-18 0.5-1 0.2-0.4 0.1 0-5 surplus
Style keeping liquid (setting lotion)
Composition Example ％
Solvent film forming component softening agent conditioning agent antistatic additive emulsifying agent aromatic dyestuff enzyme water Isopropyl alcohol vinyl pyrrolidone/vinyl acetate copolymer vinyl pyrrolidone/dimethyl aminoethyl methacrylate protolysate cetyl trimethyl ammonium chloride ethoxylated castor oil lipase 12-20 2-3.5 0.2-1 0.2-0.5 0.1-0.5 0.1-0.5 0.1-0.2＜0.1 0-5 surplus
Food and feed
In addition, conjugated enzyme or the polypeptide with the allergenicity that weakens of the present invention also can be used for producing food and feed.
Proteolytic enzyme
Gluten in the whole meal flour is the necessary composition that decision flour is used to the ability in the bakery product.Gluten phase modification for dough needs proteolytic ferment sometimes, and is for example, with proteolytic enzyme that flint wheat flour is softening.
Neutrase Be a kind of commercially available neutral metal proteolytic enzyme, can use it for and guarantee uniform dough quality and bread quality, and improve local flavor.Gluten moderately or more up hill and dale is degraded into peptide, therefore,, must carries out strictness control for fear of overbating of dough.
Also proteolytic enzyme can be used for milk-protein is carried out modification.
When producing cheese,, can use proteolytic enzyme such as rennet or rennin in order to allow the casein coagulation in Ruzhong.
In wine-making industry, proteolytic enzyme is used for the cereal that brew do not germinate and controls nitrogen content.
In the animal-feed goods, proteolytic enzyme is used to enlarge the Digestive tract of animal.
Lipase
It is quite novel things that lipase is applied to bake industry.Add lipase and can improve the characteristic of dough, and improve the bread quality of production, be embodied in bread crumb structure with bigger volume, improvement and the bread crumb color of Geng Bai.Viewed effect can be interpreted as a kind of like this mechanism: wherein, with face during, lipase has changed the interaction between gluten and some lipid fragments.Can produce the gluten structure of improvement like this.
Belong to some mozzarella type brown (blue roan) cheese (as, Danablue) and other local flavor that contains the milk-product of dairy fats form and to depend on that dairy fats is degraded to free lipid acid.Lipase can be used to produce the local flavor of this based article.
Produce in the industry in oils and fat, lipase is used to such as the purpose that reduces undesirable byproduct, so that fat is carried out modification by transesterify, and synthetic ester.
Oxydo-reductase
In addition, the oxydo-reductase of the present invention with the allergenicity that weakens can be used for producing food and feed.
Be applicable to that several oxydo-reductase that bake are glucose oxidase, lipoxygenase, peroxidase, catalase and combination thereof.Usually, the baker strengthens gluten by adding xitix and potassium bromate.Some oxydo-reductase can be used for substituting the bromate of dough system, method is by the sulfydryl in the oxidation gluten.Thereby the formation disulfide linkage causes the formation of more resilient dough stronger, that have bigger resistance.
Gluzyme TM(NOVO Nordisk A/S) is the glucose oxidase preparation with catalase activity, can use it for alternative bromate.Dough strength is by bigger anti-mechanical impact force, better toasts swelling property and bigger indexs such as loaf volume are weighed.
Carbohydrase
The difference of amylase content can cause baking the difference of quality in the flour.In order to make flour unified, must add amylase.Amylase and pentosanase can be provided for yeast-leavened sugar usually, improve loaf volume, and delaying retrogradation reduces the viscosity that becomes Chen Sudu and formed by piperylene glue.To provide the example of carbohydrase below.
Some living wheat starch enzyme can be used for the shelf-life of bread is prolonged 2 or 3 days, and can not cause the viscosity of product.By non-reducing end cracking, pasted starch is carried out the selectivity modification from starch molecule, low molecular weight sugar and dextrin.The possibility of the starch generation solarization of modification is less by this way.The lower molecular weight that is produced is the moisture holding capacity that carbohydrate can improve bakery product, and can not produce the moderate-length dextrin of the tackiness that can cause in the finished product.Described enzyme is by inactivation during bread baking, and therefore, this enzyme can be regarded as the processing material that needn't mark on label.Almost can get rid of the excessive of Novamyl.
Can improve loaf volume with fungal alpha-amylase, it can further improve bread crumb structure well and uniformly.Described α-Dian Fenmei is the endoenzyme that can produce maltose, dextrin and glucose.The temperature that is higher than starch gelatinization temperature can make cereal and some bacterial inactivation, and therefore, it can cause less loaf volume and viscosity bread inside in the time of in adding wheat dough.Fungamyl has heat labile advantage, and when just being lower than gelatinization point inactivation.
Contain the active zymin of some kinds of pentosanases and hemicellulose and can improve the processing and the stability of dough, and improve its freshness, bread crumb structure and loaf volume.
By the piperylene part in the hydrolysis flour, can make it lose most of water binding, this part water just can use for starch and gluten.Described gluten becomes more flexibility and extendability, and described starch is become be easier to gelatinization.Pentosanase and emulsifier combination can be used or substitute emulsifying agent.
In addition, carbohydrase can be used for by the Starch Production syrup, and it is widely used in soft drink, sweet food, meat product, milk-product, ice-creams, infant food, jam etc.
The conversion of starch normally divided for three steps carried out.At first, make starch liquefacation with α-Dian Fenmei.Obtain the main Star Dri 5 that constitutes by oligosaccharides and dextrin.
Handle described mixture with amyloglucosidase then, so that described oligosaccharides is become glucose with dextrin hydrolysis.Can obtain increasing the sweet food product like this.High malt sugar syrup if desired can use beta-amylase separately or uses with Starch debranching enzyme (debranching factor).
Can make the glucose mixture become sweeter by glucose mixture being isomerizated into fructose.
In sugar-refining industry, the decomposition of quickening starch in the sugarcane juice is the measure of using always.Reduce contents of starch in the raw sugar thus, and help the filtration carried out in refined sugar factory.
In addition, dextranase can be used for decomposing the dextran of crude sugar juices and syrup.
In system alcohol industry, α-Dian Fenmei is applicable to the starch in the dilution distillatory fermented liquid.
In wine-making industry, α-Dian Fenmei is used for promoting liquefaction.
In Dairy industry, beta-galactosidase enzymes (Sumylact L) can be used to produce diabetic milk, use for the patient who suffers from lactose malabsorption.
When using the breast of handling through Sumylact L to produce local flavor milk system beverage, can reduce the addition of sugar, but can not reduce the sugariness of these goods.
When producing condensed milk, can avoid lactose crystnization by the Sumylact L processing, and therefore reduce the danger of the multiviscosisty that causes because of casein coagulation in the lactose crystn.
When using the breast of handling through Sumylact L (or whey) to produce ice-creams, can not produce lactose crystn, and defective, sand grains can not occur.
In addition, as disclosed in the WO 94/21785 (Novo Nordisk A/S).Known zytase can be used for some foods/feeds industry purposes.
α-Dian Fenmei is used for the animal-feed industry,, improves the digestibility of starch so that it is added in the feed that contains cereal.
Antimicrobial polypeptide
In meat processing industry, some bacterial degradation enzyme can be used for such as the purpose (referring to U.S. Pat 5,354,681, Novo Industri A/S) of cleaning trunk.
Transferring enzyme
Trans-glutaminases with the allergenicity that weakens of the present invention is applicable to produces food and feed.
Trans-glutaminases has the ability of crosslinking protein.
Above-mentioned characteristic can be used for gelling and contain proteic water.Can use it for and produce spread food (Danish Patent Application of Novo Nordisk A/S number 1071/84).
Trans-glutaminases can be used to improve the baking properties of flour, for example, use it for and make cake, as local flavor, mouthful strength, mouthfeel and the bigger volume (referring to JP 1-110147) that improves with improved characteristic by whole meal flour being carried out modification.
In addition, can be used for producing and stick with paste the based food material, as being used as such as ice-creams, cake surface decoration (toppings), frozen food confection, mayonnaise and lower fat spread food (referring to WO93/22930, Novo Nordisk A/S).
In addition, can by breast and milk product make be used for cultured milk, wood this, the gel of cheese, pudding, orange juice, and combine with meat mincing are last, improve the taste and the structure (referring to WO94/21120 and WO 94/21129, Novo, Nordisk A/S) of food protein.
Phytase
Phytase of the present invention is applicable to production food, as breakfast cereal, cake, sweet food, beverage, bread or soup etc., and animal-feed.
Phytase can be used for utilizing in the plant protein source institute's bonded phosphorus in existing myo-Inositol hexaphosphate/phytinic acid, or is used for utilizing the mineral substance that is incorporated into the important nutritive value of having of phytinic acid mixture.
The phytase of microorganism can be added in the feed of monogastric animal, in order to avoid be that this feed adds inorganic phosphorus (referring to US patent 3,297,548).
Also phytase can be used for soyabean processing.Soyflour can contain high-load antinutritional factor myo-Inositol hexaphosphate, it makes this protein source not be suitable for the feed of infant food and fish, calf and other non-ruminant animal, because myo-Inositol hexaphosphate can the wherein existing essential minerals of chelating (referring to EP 0 420 358).
Phytase can be used for toasting purpose equally, by the dough that contains whole meal flour etc. and phytase that baking is cut apart, can make the bread (referring to JP-0-3076529-A) with better quality.
Known aspergillus with high phytase activity can be used for producing refining Japanese sake (referring to JP-0-6070749-A).
Fabric applications
Proteolytic enzyme
Proteolytic enzyme can be used for coming unstuck of silk and sand washing.
Lipase
During the fabric finishing, can remove the lipid material (for example, triglyceride level) contain hydrophobic ester (for example, referring to WO 93/13256, Novo Nordisk A/S) with lipase.
Oxydo-reductase
When the bleaching cleaning fabric, can remove excessive hydrogen peroxide with catalase.
Carbohydrase
Cellulolytic enzyme is widely used in finishing denim clothes, so that the color density generation localized variation of fabric (enzymatic " granite-wash ").
Also cellulolytic enzyme can be used for biopolishing technology.Biopolishing technology is a kind of special processing to yam surface, and it can improve the quality of fabric processing and outward appearance aspect, and can not lose the wetting properties of fabric.Can be with carrying out biopolishing such as the method that is disclosed among the WO 93/20278.
In the process of textile fabric, yarn will bear sizable mechanical tension.In order to prevent its fracture, to be coated with usually with (starching) colloid material (slurry) with its reinforcement.Modal sizing agent is natural or the starch of modified form.Only (being destarch) just can realize even and persistent finishing after the slurry on removing fabric.Preferably with the destarch of amylolytic enzyme realization with the fabric of the slurry sizing that contains starch or treated starch.
Oral and externally applied medicine
Proteolytic enzyme
Can (for example, trypsinase and Quimotrase) various combination be used for oral pharmaceutical and externally applied medicine, with treatment such as the disease of inflammation, oedema and damage with highly purified proteolytic enzyme.
Leather is produced
Transferring enzyme
Known trans-glutaminases can be used as the casein finishing (referring to WO94/13839) that stiffening agent is used for leather.
Hard surface cleaning
With the grocery trade is example, and the cleaning of crust is difficulty normally, as be used to produce dairy products, the equipment of meat, sea-food, beverage etc., have complicated shape usually.Confirmed already that the surfactant composition with gel that contains enzyme and form of foam can promote and improve the cleaning of crust.Be suitable for adding enzyme particularly proteolytic enzyme, lipase, amylase and cellulase in the described surfactant composition.
The described hard-surface cleaning composition that contains enzyme also is applicable to transportation means, for example, is used to clean the cleaning of automobile and general vehicle.
At last, the present invention relates to the application in containing the goods of polypeptide of the conjugate of the present invention or the present composition.
At first, conjugate of the present invention or composition can be used for personal-care supplies, as hair care and hair treatment goods.Comprising for example following products: shampoo, face cream, hair conditioner, hair-waving composition, composition for hair dying, hair tonic, lotion, hair-cream, shampoo, hair moistening liquid and hair spray.
Other purposes is the mouth care goods, as tooth powder, collutory, chewing gum.
Also relate to skin care implement and makeup, as (make-up base), face powder, whitening powder at the bottom of protective skin cream, skin care breast, cleaning frost, scavenging solution, cleaning breast, cold cream, soap frost, nutrition essence, skin care washing lotion, milky lotion, calamine lotion, hand lotion, soap powder, transparent soap, sunblock lotion, sun-screening agent, shaving foam, shaving cream, oil for baby, lipstick, lipstick, milkiness foundation cream (creamy foundation), face powder, eye shadow powder, powder, foundation cream, the cosmetic.
Conjugate of the present invention also is applicable to the contact lens sanitary product.Described articles for use comprise that contact lens clean and sterilizing article.
Also relate to such as the application in cleaning powder, soap, soap bar, the liquid soap.
Material and method
Enzyme
Come from the purifying peroxidase monomer (Mr=39kDa) (available from Novo Noridsk A/S) of wild-type Coprinus cinereus.
Lipase: Lipolase (available from Novo Noridsk A/S).
Cellulase: according to Boisset, the Care zyme of described method such as C. (FEBS communication, 376, p.49-52, (1995)) preparation Core.
Dextran-peroxidase A (by the Kem-En-Tech preparation of Denmark).
Dextran-peroxidase B (by the Kem-En-Tech preparation of Denmark).
Dextran-cellulase (by the Kem-En-Tech preparation of Denmark).
Dextran-lipase (by the Kem-En-Tech preparation of Denmark).
Solution
PBS Tween 20 Ausubel, F.M. etc. (editor), 1994
The anti-goat of rabbit (Sigma A-4187)
Alkaline phosphatase damping fluid (pH=9.0)
NaCl 5.844g
MgCl 2，6H 2O 1.02g
Diethanolamine 10.51g
With HCl pH is transferred to 9.0, and add Milli-Q water to 1 and rise volume.
Stop buffer
EDTA, disodium 74.44g
K 2HPO 4 174.2g
NAH 3 0.2g
Add Milli-Q water to 1 liter, transfer pH to 10 with about 22.5g KOH.
Experimental animal
Dunkin Hartley cavy is (available from Charles River, DE)
Equipment
ELISA reader: Ceres 900 HDi
Preparation antilipase and anti-cellulase, antiperoxidase
Use white new zealand rabbit.Every kind of antigen inoculation 2-4 rabbit.Use FCI (Freund's incomplete adjuvant).Every rabbit of per injection is used 50-100 μ g zymoprotein.For the manufacture order specific antibody, use the very high antigen of purity.
At its neck back side is that every rabbit is injected freshly prepd 1: 1 antigen of (subcutaneous) 1ml and the stabilized mixture of adjuvant.
Injection once injected for 10 weeks weekly.
Gather a blood sample from described rabbit after the 5th immunization, and at Quchtterrlony check (Nils H Axelsen, (1983), " gel immunoprecipitation technical manual ", Blackwell science and technology press) when its titre reaches 1/18-1/32, extract the intravital blood of described rabbit, and antibody purification.
Be used for determining IgG 1The ELISA method of positive cavy
Use the anti-cellulase of rabbit antiperoxidase 1: 8000, rabbit 1: 8000 and 1: 6000 pair of ELISA microtiter plate of rabbit antilipase in the carbonate buffer solution to apply respectively, and 4 ℃ of following overnight incubation.Inferior daily 2% BSA carries out sealing in 1 hour to described plate, and washs 3 times with PBSTween 20.
Peroxidase, cellulase Core and lipase are added on the relevant plate, and its concentration is 1 μ g zymoprotein/ml.
All guinea pig serum samples are added on the elisa plate, peroxidase is used 10 μ l serum and 90 μ l PBS, and 1: 50 serum dilution is used for cellulase and lipase, cultivated 1 hour, and wash 3 times with PBS Tween 20.
Then with the anti-cavy IgG of goat 1(Nordic immunology 44-682) adds on the described titer plate with 1: 4000 PBS damping fluid of 0.5% BSA, cultivates 1 hour, and washs with PBSTween 20.
Add the alkaline phosphatase with the anti-goat mark of 1: 8000 rabbit, and cultivated 1 hour, washing is 2 times in PBS Tween 20, and washs 1 time with diethanolamine.
Under 37 ℃, launch the alkaline phosphatase 30 minutes that described mark crosses with p-nitrophenyl phosphate, stop said process with the calcium that contains EDTA/sodium damping fluid (pH10), and with the ELISA reader at 405/650 time reading of OD.
On all elisa plates, all contain double blind trial.
Positive and negative serum value adds that with average blind test value 2 times of standard deviations obtain.The tolerance range that obtains thus is 95%.
Described test has explanation more completely in APR 95255001, ED-9516670, these data can be asked for to Novo Nordisk A/S.
Embodiment
Example 1
Test with the allergenicity that the peroxidase of modifying carries out
According to the explanation among the ED-9516670 (can ask for), handle Dunkin Hartley cavy with the purifying monomer (cavy 21-30) of the Coprinus cinereus peroxidase of 1.0 μ g purifying and the dextran-peroxidase A (cavy 31-40) and the dextran-peroxidase B (cavy 41-50) of 1.0 μ g modification by dosage in the tracheae to Novo Nordisk A/S.
In 8 time-of-weeks with the IgG of above-mentioned all cavys of ELISA method test 1Output (expression transformation reactions).
Fig. 1 is illustrated in described duration of test and shows as IgG 1Male Dunkin Hartley cavy quantity.
As seen from Figure 1, in any time of duration of test IgG1 male cavy quantity, the monomer peroxidase all reduces for dextran-peroxidase A and B relatively.This shows, by combining with suitable polymer support molecule such as dextran, can weaken the allergenicity of polypeptide.
Example 2
Test with the allergenicity that the lipase of modifying carries out
Repetition is in the test described in the example 1, and different is, stimulates Dunkin Hartley cavy by method in the tracheae with the modification lipase of 1 μ g purifying lipase or 1 μ g purifying.
In 10 time-of-weeks, with the IgG of above-mentioned all cavys of ELISA method test 1Output (indication transformation reactions).
As shown in Figure 2, the lipase of described modification has the allergenicity that weakens.
Example 3
Test with the allergenicity that the cellulase of modifying carries out
Repetition is in the test disclosed in the example 1, and different is to stimulate Dunkin Hartley cavy by method in the tracheae with the cellulase of 1 μ g purifying or the using modified cellulase of 1 μ g purifying.
The IgG that in 10 time-of-weeks, checks all cavys to produce with above-mentioned ELISA method 1(indication transformation reactions).
As shown in Figure 3, the lipase of modification has the sex change originality that weakens.
Can understand by above explanation those skilled in the art, under the prerequisite that does not exceed design of the present invention or scope, when enforcement is of the present invention, can make multiple changes and improvements.Therefore, scope of the present invention is understood that to be limited by the essence of following claim.

Claims (39)

1. conjugate with the allergenicity that weakens, comprise a polymer support molecule with two or more peptide molecules that are attached thereto, described polymer support molecule is selected from star PEGs, branching PEGs, polyvinyl alcohol (PVA), poly carboxylic acid, poly--(vinyl pyrrolidone) and poly--D, L-amino acid.
2. conjugate as claimed in claim 1 comprises the peptide molecule that directly is connected with described polymer support molecule.
3. conjugate as claimed in claim 2 comprises the peptide molecule that is connected with described polymer support molecule by a linkers.
4. conjugate as claimed in claim 3, comprise a polymer support molecule, have two or more peptide molecules that are attached thereto by covalent linkage on this polymer support molecule, described covalent linkage is formed between in two vinyl of divinylsulfone one.
5. as each conjugate among the claim 1-4, wherein, described polypeptide is a kind of antimicrobial polypeptide.
6. conjugate as claimed in claim 5, wherein, described polypeptide has biologic activity.
7. conjugate as claimed in claim 5, wherein, described polypeptide is a kind of enzyme.
8. conjugate as claimed in claim 7, wherein, described enzyme is proteolytic enzyme, lipase, transferring enzyme, carbohydrase, oxydo-reductase or phytase.
9. conjugate as claimed in claim 7, wherein, the molecular weight of described enzyme (Mr) is 4-200kDa.
10. as each conjugate among the claim 1-4, wherein, the molecular weight of described polymer support molecule is 1-10,000kDa.
11. as each conjugate among the claim 1-4, the wherein said total molecular weight (Mr) of puting together molecule is 50-40,000kDa.
12. as the conjugate of claim 1-4, wherein, each polymer support combines with 2-60 peptide molecule.
13. as the conjugate of claim 1-4, wherein, the peptide molecule combination that each polymer support molecule is different with two or more.
14. as the conjugate of claim 13, wherein, described polypeptide shows two kinds of different activity at least.
15., comprise one or several enzyme molecule and one or several ligand molecular as the conjugate of claim 13.
16., comprise one or several antibody molecule and one or several inhibitor molecules as the conjugate of claim 13.
17., comprise one or several acceptor molecule and one or several antibody molecule as the conjugate of claim 13.
18. a production has the method for the peptide molecule of the allergenicity that weakens, and may further comprise the steps:
I) by connect a last active part activate a kind of polymer support molecule and
Ii) under the condition that is suitable for puting together, allow two or more peptide molecules and the molecular reaction of described activated polymer support and
Iii) seal the residual activity group on the described conjugate.
19. as the method for claim 18, wherein, step I) activation of polymer support molecule is to realize by a covalently bound last active part that comes from divinylsulfone described in.
20. as the method for claim 18 or 19, wherein, described peptide molecule is each described molecule among the claim 5-9.
21. as the method for claim 18 or 19, wherein said polymer support molecule is by the amino (NH on the described peptide molecule 2), hydroxyl (OH), the carboxylic acid group (COOH) or sulfydryl be connected with this peptide molecule.
22. a composition comprises among the claim 1-17 each conjugate.
23. composition as claim 22, wherein, said composition also comprises polypeptide, albumen and/or enzyme and/or is usually used in composition in the following material: washing composition, household supplies, agrochemicals, personal-care supplies, makeup, cosmetics, medicine is handled the composition that fabric is used, composition that the cleaning crust is used and the composition that is used to produce food and feed.
24. with each describedly puts together the purposes that molecule or claim 22 or 23 described compositions are used to weaken the allergenicity of mechanicals among the claim 1-17.
25. as the purposes of claim 24, wherein, described mechanicals is personal-care supplies.
26., use it in the goods of hair care or hair treatment as the purposes of claim 25.
27., use it in the mouth care goods as the purposes of claim 25.
28., use it in tooth powder, collutory and the chewing gum as the purposes of claim 27.
29., use it in the skin care goods as the purposes of claim 25.
30., use it in the makeup as the purposes of claim 25.
31., use it for the contact lens hygienic articles as the purposes of claim 25.
32., use it in the washing composition as the purposes of claim 24.
33., use it in the liquid washing agent as the purposes of claim 32.
34., use it for and wash in the dish washing composition as the purposes of claim 32.
35., use it in soap, soap bar, the liquid soap as the purposes of claim 32.
36., use it in the oral and externally applied medicine as the purposes of claim 24.
37., use it in food or the feed as the purposes of claim 24.
38., use it in the goods of handling fabric as the purposes of claim 24.
39., use it in the composition that cleans crust as the purposes of claim 24.
CNB971922942A 1996-02-15 1997-02-07 Conjugation of polypeptides Expired - Fee Related CN1273589C (en)

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